The synthesis of poly(A) tracts having 5, 18, 31, 41, 59, 73, 100 and 150 residues, covalently attached to the 3' termini of E. coli 5S (3H) rRNA (5S rRNA-(A)n) was carried out with polynucleotide phosphorylase. These polyadenylated RNAs and unmodified 5S rRNA were utilized as substrates for ribonucleases from Citrobacter, Enterobacter, bovine pancreas, human spleen and human plasma. In all cases, short lengths of poly(A) (n equals 18) induce a powerful inhibition (38-87%) of RNase activity, which increases as the poly(A) tract is lengthened. The poly(A) mediated protection of substrate from degradation extends to RNA molecules which are not covalently linked to poly(A), demonstrating that a sub-population of poly(A) containing RNA molecules can contribute to the stability of other RNAs by inhibiting the RNase mediated hydrolysis of these substrates. The structure of 5S rRNA (Tm equals 61 degrees C) is altered upon covalent attachment of poly(A) (Tm equals 46 degrees C if 31 adenylic acid residues are present at the 3' termini). Thus, under certain conditions, 5S rRNA (A)n behaves quite differently from a mixture of 5S rRNA and poly(A). Covalent attachment of poly(A) results in a better inhibitor of RNase activity than free poly(A)184. Furthermore, if inhibition is reversed, 5S rRNA(A)n is hydrolyzed more rapidly by RNase than 5S rRNA plus poly(A).